bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.044313; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Comparative RNA-Seq Analyses of Drosophila Plasmatocytes Reveal Gene 2 Specific Signatures In Response To Clean Injury And Septic Injury 3 4 Elodie Ramond1#*, Jan Paul Dudzic1#* and Bruno Lemaitre1* 5 # Contributed equally to this work 6 1. Global Health Institute, School of Life Science, École Polytechnique Fédérale 7 de Lausanne (EPFL), Lausanne, Switzerland. 8 9 *Corresponding authors: 10 [email protected] 11 [email protected] 12 [email protected] (lead contact) 13 14 Abstract 15 Drosophila melanogaster’s blood cells (hemocytes) play essential roles in wound healing and 16 are involved in clearing microbial infections. Here, we report the transcriptional changes of 17 larval plasmatocytes after clean injury or infection with the Gram-negative bacterium 18 Escherichia coli or the Gram-positive bacterium Staphylococcus aureus compared to 19 hemocytes recovered from unchallenged larvae via RNA-Sequencing. This study reveals 676 20 differentially expressed genes (DEGs) in hemocytes from clean injury samples compared to 21 unchallenged samples, and 235 and 184 DEGs in E. coli and S. aureus samples respectively 22 compared to clean injury samples. The clean injury samples showed enriched DEGs for 23 immunity, clotting, cytoskeleton, cell migration, hemocyte differentiation, and indicated a 24 metabolic reprogramming to aerobic glycolysis, a well-defined metabolic adaptation observed 25 in mammalian macrophages. Microbial infections trigger significant transcription of immune 26 genes, with significant differences between the E. coli and S. aureus samples suggesting that 27 hemocytes have the ability to engage various programs upon infection. Collectively, our data 28 bring new insights on Drosophila hemocyte function and open the route to post-genomic 29 functional analysis of the cellular immune response. 30 31 32 33 Keywords: Drosophila, hemocytes, wounding, Staphylococcus aureus, Escherichia coli, 34 RNA sequencing, transcriptome, immunity bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.044313; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 35 Introduction 36 37 Drosophila blood cells, also called hemocytes, contribute to the cellular immune 38 response by engulfing bacteria, combatting parasites and secreting antimicrobial and clotting 39 factors. They also participate in regulating the immune response by secreting cytokines such 40 as the JAK-STAT ligands Unpaired (Morin-Poulard et al. 2013) or the Toll ligand Spätzle 41 (Irving et al. 2005; Agaisse et al. 2003; Wang et al. 2014). Hemocytes are also involved in 42 wound healing notably through the engulfment of apoptotic cells and cellular debris, the 43 stimulation of stem cell proliferation, and deposition of extracellular matrix (Evans and Wood 44 2014; Chakrabarti et al. 2016a; Arefin et al. 2014; Sánchez-Sánchez et al. 2017). Furthermore, 45 hemocytes produce enzymes essential to the melanization reaction (Binggeli 2014, Dudzic 46 2015). Recent evidence shows that Drosophila blood cells contribute not only to immunity 47 and wound healing, but are also central to host metabolism (Dolezal et al. 2019; Shin et al. 48 2020; Green et al. 2018; Mihajlovic et al. 2019). That an excessive number of hemocytes can 49 be detrimental to flies raised on a poor diet shows that hemocyte number must be tightly 50 regulated (Ramond et al. 2020). Thus, there is a current effort to better characterize the role of 51 hemocytes during the life cycle of flies. 52 Hematopoiesis occurs in several waves throughout the Drosophila life cycle. The first phase 53 of hematopoiesis establishes a pool of hemocytes from the embryonic head mesoderm. These 54 cells contribute to embryonic development by phagocytosing apoptotic cells, and through the 55 deposition of extracellular matrix (Sánchez-Sánchez et al. 2017). These embryonic derived 56 hemocytes persist in larvae, where they are subjected to several rounds of division reaching 57 about 6000 hemocytes at the end of the third instar larval stage (Holz et al. 2003). Peripheral 58 larval hemocytes are found either (i) in circulation in hemolymph or (ii) in sessile patches 59 (Lanot et al. 2001; Evans et al. 2003; Jung et al. 2005; Crozatier and Meister 2007; Honti et 60 al. 2010; Makhijani et al. 2011; Makhijani and Brückner 2012). Sessile hemocytes are 61 attached to the internal surface of the larval body wall, forming patches, some of which are 62 closely associated with secretory cells called oenocytes, as well as the endings of peripheral 63 neurons (Makhijani et al. 2011; Makki et al. 2014). Hemocytes are continuously exchanged 64 between sessile patches and circulation (Babcock et al. 2008; Welman et al. 2010). The 65 function of sessile hemocyte patches is not yet established but it has been proposed that they 66 form a diffuse hematopoietic organ (Márkus et al. 2009; Makhijani et al. 2011; Leitão and 67 Sucena 2015). Larvae also possess a special hematopoietic organ, the lymph gland, that 68 functions as a reservoir releasing hemocytes at the pupal stage or after parasitic infection. 69 Both lymph gland and embryonic derived hemocyte populations contribute to the pool of bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.044313; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 70 adult hemocytes that will ultimately decline upon ageing. Whether active hematopoiesis 71 occurs in adults is still debated (Ghosh et al. 2015; Sanchez Bosch et al. 2019). 72 Most studies on the cellular immune response focus on third instar larval hemocytes as both 73 sessile and circulating hemocytes can easily be collected and FACS sorted. Drosophila larvae 74 have two types of hemocytes in the unchallenged state: plasmatocytes, which are 75 macrophage-like, and crystal cells, rounded hemocytes which contain crystals of 76 prophenoloxidases, the zymogen form of phenoloxidases that catalyzes the melanization 77 reaction against parasites or septic injury (Rizki et al. 1980; Lanot et al. 2001; Binggeli et al. 78 2014). A third type of hemocytes, the lamellocytes, is restricted to the larval stage and 79 originates either from progenitors in the lymph gland or in periphery by transdifferentiation of 80 plasmatocytes or circulating progenitors (Rizki and RIZKI 1992; Vlisidou and Wood 2015). 81 These cells differentiate upon parasitoid wasp infestation and contribute to the encapsulation 82 and melanization of larger parasites. At the larval stage, plasmatocytes represent the most 83 abundant fraction of Drosophila blood cells (i.e. 90-95%) (Tepass et al. 1994) and express 84 several markers such as the clotting factor Hemolectin (Hml), or the phagocytic receptors 85 Nimrod C1 (NimC1 or P1) or Eater (Evans et al. 2003). The other 5-10% larval hemocytes 86 are Lozenge (Lz) positive crystal cells (Lanot et al. 2001). Only rarely can lamellocytes be 87 observed in the unchallenged larvae as these cells are induced upon wasp infestation or injury 88 (Rizki & Rizki 1992). 89 Until recently, there have been surprisingly few studies analyzing the hemocyte 90 transcriptome, possibly due to difficulties in collect enough materials. The most 91 comprehensive genome wide analysis was a characterization of whole larval hemocyte 92 populations by Irving et al. in 2005, using an Affymetrix based oligonucleotide array (Irving 93 et al. 2005). Of the 13 000 genes (total number of genes >17 500) represented in this 94 microarray, they were able to identify 2500 with significantly enriched expression in 95 hemocytes, notably genes encoding integrins, peptidoglycan recognition proteins (PGRPs), 96 scavenger receptors, lectins, cell adhesion molecules and serine proteases. Interestingly, 97 several single cell transcriptomic analyses have revealed the degree of heterogeneity of 98 Drosophila hemocyte populations, but they did not characterize the full repertoire of genes 99 expressed in hemocytes (Cattenoz et al. 2020; Tattikota et al. 2019; Fu et al.; Cho et al. 2020). 100 To better characterize the transcriptome of hemocytes, we have performed an RNAseq 101 transcriptome analysis of FACS sorted Hml positive cells. The transcriptome of Hml positive 102 (Hml+) plasmatocytes was determined in an unchallenged condition and 45 minutes 103 following clean or sceptic injury with Staphylococcus aureus or Escherichia coli. 104 Comparative transcriptomics allowed us to identify a set of genes specific to plasmatocytes in bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.044313; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 105 unchallenged or challenged condition, revealing the various contributions of these cells to 106 host defense, wound healing and metabolism. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.16.044313; this version posted April 18, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 107 Results 108 109 Study design 110 We performed RNA sequencing of mRNA to analyze the global gene expression profile 111 changes of Drosophila hemocytes from third instar larvae either unchallenged or collected 45 112 minutes after clean injury or septic injury with a needle dipped in concentrated bacterial 113 pellets of Staphylococcus aureus or Escherichia coli. To isolate the plasmatocytes from other 114 unwanted cells of the hemolymph preparation, we used the HmlΔ.Ds-Red.nls fluorescent 115 marker, which is specifically expressed in most plasmatocytes, and to a lesser extent in newly 116 differentiated crystal cells (Goto et al.